MARCH8/NSUN6/ROS-mediated DNA damage positive feedback loop regulates cisplatin resistance in osteosarcoma.

Osteosarcoma is the most common primary malignant bone tumor in children and adolescents and is often characterized by resistance to chemotherapy. Although RNA 5‑methylcytosine (m5C) modification is known to contribute to tumor progression, its exact role in osteosarcoma drug resistance remains poorly understood. Here, we identify NOP2/Sun RNA methyltransferase family member 6 (NSUN6) as an m5C methyltransferase that positively correlates with osteosarcoma progression. Mechanistically, the E3 ubiquitin ligase membrane‑associated RING‑CH‑type finger 8 (MARCH8) ubiquitinates NSUN6 at Lys271 and Lys462, leading to its proteasomal degradation. Reduced NSUN6 expression lowers m5C modification on peroxisomal biogenesis factor 1 (PEX1) and peroxisomal biogenesis factor 3 (PEX3) mRNAs, destabilizing them through loss of binding by the m5C reader YBX1. In turn, this downregulates peroxisome synthesis and catalase (CAT) protein production, causing increased intracellular reactive oxygen species (ROS), DNA damage, and heightened sensitivity of osteosarcoma cells to cisplatin. Furthermore, elevated ROS levels reinforce NSUN6 ubiquitination and degradation by enhancing the NSUN6-MARCH8 interaction, establishing a positive feedback loop. Collectively, these findings highlight an intricate NSUN6-m5C-YBX1-PEXs signaling axis that governs peroxisome biogenesis, ROS accumulation, and cisplatin responsiveness in osteosarcoma. Our work not only clarifies the role of m5C in osteosarcoma drug resistance but also offers a potential therapeutic angle for targeting NSUN6 and its peroxisome‑regulating network to overcome chemoresistance.